Comparison of Phenolic Compounds, Carotenoids, Amino ......foods Article Comparison of Phenolic Compounds, Carotenoids, Amino Acid Composition, In Vitro Antioxidant and Anti-Diabetic

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Comparison of Phenolic Compounds, Carotenoids,Amino Acid Composition, In Vitro Antioxidant andAnti-Diabetic Activities in the Leaves of SevenCowpea (Vigna unguiculata) Cultivars

Mapula R. Moloto 1 , Anh Dao T. Phan 2 , Jerry L. Shai 3 , Yasmina Sultanbawa 2 andDharini Sivakumar 1,2,*

1 Phytochemical Food Network Research Group, Department of Crop Sciences, Tshwane University ofTechnology, Pretoria West 0001, South Africa; [email protected]

2 ARC Industrial Transformation Training Centre for Uniquely Australian Foods, Queensland Alliance forAgriculture and Food Innovation, The University of Queensland, St. Lucia QLD 4108, Australia;[email protected] (A.D.T.P.); [email protected] (Y.S.)

3 Department of Biomedical Sciences, Tshwane University of Technology, Arcadia, Pretoria 0001, South Africa;[email protected]

* Correspondence: [email protected]

Received: 5 August 2020; Accepted: 5 September 2020; Published: 12 September 2020��

Abstract: Cowpea is a well-known nutrition rich African leafy vegetable that has potential to sustainfood and nutrition insecurity in sub-Saharan Africa. Consumption of cowpea legumes is associatedwith reduced risk of type 2 diabetes mellitus. Therefore, the present study was designed to evaluatethe (i) variation in phenolic metabolites in seven cowpea cultivars (VOP1, VOP2, VOP3, VOP4, VOP5,VOP7, and VOP8 using UHPLC coupled with high resolution Q-TOF-MS technique, (ii) in vitroantioxidant activity using ferric reducing/antioxidant capacity (FRAP) assay (iii) in vitro anti-diabeticeffects and (iv) composition of carotenoids and amino acids of theses cowpea cultivars. The resultsof this study demonstrated that gentisic acid 5-O-glucoside, quercetin 3-(2G-xylosylrutinoside)and Quercetin 3-glucosyl-(1->2)-galactoside were highest in VOP1 VOP4 and VOP5, respectively.High inhibition (>50%) of α-glucosidase and α-amylase activities was shown by the leaf extracts(50 and 25 mg/mL) of VOP1 and VOP4. Cowpea cultivars VOP1 and VOP4 demonstrated the highestgene expression levels of regulation of glucose transporter GLUT4 in C2C12 skeletal muscle cells,similar to insulin. A positive correlation exited between the phenolic components and the inhibitoryeffect of antidiabetic enzymes and FRAP activity. Cytotoxic effect was not detected in vitro in anycowpea cultivar. Lutein (124.6 mg/100 g) and all-trans-beta-carotene (92.6 mg/100 g) levels werehighest in VOP2 and VOP1, respectively. Cowpea cultivars VOP3 and VOP4 showed potential to fulfilthe daily requirements of essential amino acids. Thus, based on this information, cowpea (leaves)genotypes/cultivars can be selected and propagated for the further development of supplementaryfoods or functional food ingredients.

Keywords: leafy vegetables; polyphenols; anti-diabetic enzymes; protocatechuic acid-O-glucoside;lutein; phenylalanine; antioxidant capacity; regulation of glucose transporter

1. Introduction

Cowpea (family Fabaceae) is one of the indigenous leafy vegetables that contributes significantlyto household food and nutritional security and societal health, as well as adding variety to cereal-basedstaple diets in the Southern African region [1]. Cowpea is drought tolerant and well adapted for warm

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weather conditions. Indigenous fruits and vegetables can be considered as an affordable strategy indiet diversification and the eradication of hidden hunger [2]. In addition, the inclusion of cowpealeaves in African cuisine will add more nutritional value to the consumers as they are a rich source ofprotein, functional compounds (amino acids, polyphenols, and carotenoids), vitamins (provitamin A,folate, thiamin, riboflavin, and vitamin C) and minerals (calcium, phosphorus, and iron) [3]; the proteincontent of cowpea leaves ranges from 21.5 to 43.7% [4]. The protein content in cowpea leaves arerelatively comparable with other protein-rich leafy vegetables such as spinach (38.2%), Brussels sprouts(34.1%), kale (36.8%) and mustard greens (34%) [5].

Cowpea leaves consist of protein building blocks, essential amino acids, such as isoleucine,leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and histidine [4], and thenon-essential amino acids tyrosine, aspartate, glutamate, glycine, alanine, cysteine, serine and proline [4].Research findings of Van Jaarsveld et al. [6] stated that 3/4 cup (90 g) of cowpea leaves fulfil ≥75% ofrecommended dietary allowance (RDA) for vitamin A (700–900 µg/day for adults [7]; and 25–50% RDAfor Fe (10 mg/day) for children (4–8 years) [8].

Consumption of cowpea legumes is associated with reduced risk of type 2 diabetes mellitus andobesity [3]; moreover, the dietary phenolic compounds demonstrated inactivation of carbohydratedigestive enzymes, α-amylase and β-glucosidase, and activated appropriate antihypoglycemicagents [9]. The aforementioned enzymes play a vital role in controlling blood glucose levels andobesity due to their ability to reduce the re-absorption of glucose in the intestine. Inclusion ofhigher dietary fibre and low glycaemic index foods in daily meals has been proved to reduce bloodglucose levels, thereby reducing the risk of type 2 diabetes mellitus [9]. Indigenous edible plantextracts demonstrated anti-diabetic effects [10], such as insulin (diabetic drug), by executing the vitalregulatory mechanism transporting the glucose uptake into skeletal muscle and adipose tissue byfacilitating glucose transporter protein type-4 (GLUT4), playing a major role in the management oftype 2 diabetes [11]. GLUT4 therefore plays a vital role in glucose homeostasis of skeletal muscle cellsand the removal of glucose from blood circulation [11].

The number of people affected with type 2 diabetes in Africa was projected to increase to 41.5 millionin 2035, and it will be more prevalent in people who are between the ages of 40–59 [12]. Another approachto tackle type 2 diabetes and obesity is formulating supplementary foods using indigenous plantingredients based on their nutritional values and phytochemical profiles. Implementing supplementaryfeeding programmes would be an affordable strategy and would result in lowering postprandialglycaemia at least partly by promoting skeletal muscle glucose uptake and intensifying the metabolism.

Chemometric analysis is widely used in metabolomics analysis for characterisation, and assessmentof the divisiveness in the overall bioactive metabolites of functional foods [13]. In addition, it isessential to build up a phytochemical database for bioactive compounds in foods that can link to thechemical properties associated with nutritional and nutraceutical effects [13]. Therefore, this studyaimed to (i) investigate the different phenolic metabolites in cowpea accessions using UHPLC coupledwith high resolution Q-TOF-MS technique and a chemometric analysis approach, (ii) characterise andquantify the phenolic compounds carotenoids and amino acids, (iii) determine the in vitro antioxidantcapacity and anti-diabetic activity, and (iv) understand the molecular basis for ‘insulin like’ activity ofthe polyphenols extracted from different African grown cowpea leaf cultivars, on the regulation ofglucose transporter GLUT4. This study will lead in identifying cowpea cultivars, for the developmentof supplementary foods that are a rich source of amino acids, carotenoids, phenolic compounds andantioxidant properties, and relate to the dietary roles of cowpea (leaf) functional compounds in type2 diabetes management. This approach will significantly benefit the consumers belonging to thevulnerable groups and the food manufacturers.

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2. Materials and Methods

2.1. Chemicals

Polyphenols (≥95% purity), including chlorogenic acid, catechin, luteolin, epicatechin andrutin, were purchased from Sigma-Aldrich (Johannesburg, South Africa). Carotenoids (analyticalstandards), including violaxanthin, lutein, zeaxanthin and all-trans-beta-carotene, were sourced fromSigma-Aldrich (Sydney, NSW, Australia). All the other chemicals and solvents (HPLC grade) werepurchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (Johannesburg, South Africa).

2.2. Plant Material

Seeds of seven cowpea (Vigna unguiculata L. Walp) cultivars (VOP1, VOP2, VOP3, VOP4, VOP5,VOP7, VOP8) (Figure 1) planted in Southern African region were obtained from Dr. Abe Gerrano andMs. Lindiwe Khoza from the Agricultural Research Council’s (Pretoria, South Africa). Cowpea plantswere propagated at the experimental plot at the Tshwane University of Technology (25◦43′53.55′′S,28◦09′40.38′′E, on 1230 m.a.s.l) during the summer of 2018, and the average temperature ranged from 16to 30 ◦C. The experimental unit was arranged in a completely randomised design with five replicationsfor each cowpea cultivar and the seeds planted in pots. The irrigation (100 mL/day) was kept to aminimum, as its production was recommended in dry lands. The leaves were harvested at 8-leaf stage,reached after 60 to 95 days of planting. Leaves (1.5 kg) that were free from decay, damage or soilparticles were harvested and rinsed in tap water, then snap frozen in liquid nitrogen and subsequentlystored at −80 ◦C for biochemical analysis. Another portion of leaves (150 mg) was freeze-dried (−85 ◦C,LyoQuest −55/Telstar, Shanghai, China) and ground into fine powder for carotenoid and total proteincontent and amino acid analysis.

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2. Materials and Methods

2.1. Chemicals

Polyphenols (≥95% purity), including chlorogenic acid, catechin, luteolin, epicatechin and rutin, were purchased from Sigma-Aldrich (Johannesburg, South Africa). Carotenoids (analytical standards), including violaxanthin, lutein, zeaxanthin and all-trans-beta-carotene, were sourced from Sigma-Aldrich (Sydney, NSW, Australia). All the other chemicals and solvents (HPLC grade) were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (Johannesburg, South Africa).

2.2. Plant Material

Seeds of seven cowpea (Vigna unguiculata L. Walp) cultivars (VOP1, VOP2, VOP3, VOP4, VOP5, VOP7, VOP8) (Figure 1) planted in Southern African region were obtained from Dr. Abe Gerrano and Ms. Lindiwe Khoza from the Agricultural Research Council’s (Pretoria, South Africa). Cowpea plants were propagated at the experimental plot at the Tshwane University of Technology (25°43′53.55′′ S, 28°09′40.38′’ E, on 1230 m.a.s.l) during the summer of 2018, and the average temperature ranged from 16 to 30 °C. The experimental unit was arranged in a completely randomised design with five replications for each cowpea cultivar and the seeds planted in pots. The irrigation (100 mL/day) was kept to a minimum, as its production was recommended in dry lands. The leaves were harvested at 8-leaf stage, reached after 60 to 95 days of planting. Leaves (1.5 kg) that were free from decay, damage or soil particles were harvested and rinsed in tap water, then snap frozen in liquid nitrogen and subsequently stored at −80 °C for biochemical analysis. Another portion of leaves (150 mg) was freeze-dried (−85 °C, LyoQuest −55/Telstar, Shanghai, China) and ground into fine powder for carotenoid and total protein content and amino acid analysis.

VOP3

VOP4 VOP5 VOP7

VOP1 VOP2

Figure 1. Cont.

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Figure 1. Different cowpea cultivars used in this study.

2.3. Predominant Phenolic Metabolic Profile

Phenolic compounds were extracted from cowpea leaves using the method described by Ndou et al. [14] and Managa et al. [15]. Cowpea snap frozen leaf samples (50 mg) were extracted in ethanol/water solution (70:30, v/v), ultrasonicated for 30 min then centrifuged (Hermle Z326k, Hermle Labortechnik GmbH, Wehingen, Germany) at 1000× g for 20 min at 4 °C. The supernatants were collected and filtered through a 0.22-µm polytetrafluorethylene filter prior to UPLC-QTOF/MS analysis.

Peak identities and quantification of predominant polyphenol metabolites were carried out using an UPLC-QTOF/MS system (Waters, Milford, MA, USA) equipped with a Quadrupole 120 time-of-flight (QTOF) mass spectrometer. The chromatographic conditions were performed as per Ndou et al. [14] and Managa et al. [15]. Due to the unavailability of commercial standards, these were semi-quantitatively measured against calibration curves prepared using chlorogenic acid, catechin, luteolin, epicatechin and rutin. Data processing using TargetLynx software was conducted as described previously [14,15]. The differences between the phenolic metabolic profiles of the different cowpea cultivars were analysed using an unsupervised Principal Component Analysis (PCA) approach using the data generated by the UPLC–Q-TOF/MS analysis. PCA was performed to reduce the number of variables in the data matrix in order to select the most discriminating cowpea cultivars as stated by Ndou et al. [14]. Therefore, the UPLC data were exported as an mzXML file and aligned by Marker Lynx 4.1 in the Apex Trac ™ tool and imported into SIMCA-P + 12.0 and fir the PCA analysis. However, to explain the differences between the cultivar groups and to identify the potential characteristic markers (metabolites) responsible for discrimination between the cowpea cultivars, supervised Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) was performed.

2.4. Trolox Equivalent Antioxidant Capacity (TEAC) FRAP Assay

FRAP assay was carried out according to Mpai et al. [16], using a micro-plate reader (CLARIOstar Plus BMG Labtec, Lasec, Cape Town, South Africa) and snap frozen cowpea leaf samples (0.2 g). Briefly, 15 µL aliquot of leaf extract and 220 µL of FRAP reagent solution were added to the wells. The absorbance was read at 593 nm and the standard curve of Trolox was constructed to calculate the reducing antioxidant capacity was expressed as µmol TEAC/100 g.

2.5. Antidiabetic Activity

2.5.1. α-Glucosidase Inhibition Assay

The α-glucosidase inhibitory activity was measured according to the method described by Sagbo et al. [17], using a micro-plate reader (CLARIOstar Plus BMG Labtec, Lasec, Cape Town, South Africa). Briefly, 5 µL of the leaf extract (mentioned in Materials and methods Section 2.3) of cowpea cultivars VOP1 and VOP4 (50, 25 and 5 mg/µL) was mixed with 20 µL of 50 µg/mL α-glucosidase solution into

VOP8

Figure 1. Different cowpea cultivars used in this study.

2.3. Predominant Phenolic Metabolic Profile

Phenolic compounds were extracted from cowpea leaves using the method described byNdou et al. [14] and Managa et al. [15]. Cowpea snap frozen leaf samples (50 mg) were extracted inethanol/water solution (70:30, v/v), ultrasonicated for 30 min then centrifuged (Hermle Z326k, HermleLabortechnik GmbH, Wehingen, Germany) at 1000× g for 20 min at 4 ◦C. The supernatants werecollected and filtered through a 0.22-µm polytetrafluorethylene filter prior to UPLC-QTOF/MS analysis.

Peak identities and quantification of predominant polyphenol metabolites were carried out usingan UPLC-QTOF/MS system (Waters, Milford, MA, USA) equipped with a Quadrupole 120 time-of-flight(QTOF) mass spectrometer. The chromatographic conditions were performed as per Ndou et al. [14] andManaga et al. [15]. Due to the unavailability of commercial standards, these were semi-quantitativelymeasured against calibration curves prepared using chlorogenic acid, catechin, luteolin, epicatechinand rutin. Data processing using TargetLynx software was conducted as described previously [14,15].The differences between the phenolic metabolic profiles of the different cowpea cultivars were analysedusing an unsupervised Principal Component Analysis (PCA) approach using the data generatedby the UPLC–Q-TOF/MS analysis. PCA was performed to reduce the number of variables in thedata matrix in order to select the most discriminating cowpea cultivars as stated by Ndou et al. [14].Therefore, the UPLC data were exported as an mzXML file and aligned by Marker Lynx 4.1 in theApex Trac™ tool and imported into SIMCA-P + 12.0 and fir the PCA analysis. However, to explain thedifferences between the cultivar groups and to identify the potential characteristic markers (metabolites)responsible for discrimination between the cowpea cultivars, supervised Orthogonal Projections toLatent Structures Discriminant Analysis (OPLS-DA) was performed.

2.4. Trolox Equivalent Antioxidant Capacity (TEAC) FRAP Assay

FRAP assay was carried out according to Mpai et al. [16], using a micro-plate reader (CLARIOstarPlus BMG Labtec, Lasec, Cape Town, South Africa) and snap frozen cowpea leaf samples (0.2 g).Briefly, 15 µL aliquot of leaf extract and 220 µL of FRAP reagent solution were added to the wells.The absorbance was read at 593 nm and the standard curve of Trolox was constructed to calculate thereducing antioxidant capacity was expressed as µmol TEAC/100 g.

2.5. Antidiabetic Activity

2.5.1. α-Glucosidase Inhibition Assay

The α-glucosidase inhibitory activity was measured according to the method described bySagbo et al. [17], using a micro-plate reader (CLARIOstar Plus BMG Labtec, Lasec, Cape Town,South Africa). Briefly, 5 µL of the leaf extract (mentioned in Materials and methods Section 2.3) ofcowpea cultivars VOP1 and VOP4 (50, 25 and 5 mg/µL) was mixed with 20µL of 50µg/mLα-glucosidasesolution into a well, then 60 µL of 67 mM potassium phosphate buffer (pH 6.8) was added to themixture and incubated for 5 min at 35 ◦C. Subsequently, 10 µL of 10 mM ρ-nitrophenyl-α-D-glucoside

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solution (PNPGLUC) was added and the incubation was extended for an additional 20 min at 35 ◦C,followed by adding 25 µL of 100 mM Na2CO3; the absorbance was read at 405 nm. The absorbancewas measured for the cowpea leaf extracts, or acarbose, and the blank (samples without α-glucosidase).The enzyme inhibitory activity was calculated according to Sagbo et al. [17] and expressed as thepercentage of α-glucosidase inhibition.

2.5.2. α-Amylase Inhibition Assay

The α-amylase inhibition assay was performed according to the method described bySagbo et al. [17], without any modifications, using a micro-plate reader (CLARIOstar Plus BMGLabtec, Lasec, Cape Town, South Africa) monitored at 580 nm. The enzyme inhibitory activity wasexpressed as the percentage of α-amylase inhibition.

2.5.3. Gene Expression of GLUT-4

Treatment of cells with the leaf extracts (100 µL) of cowpea cultivars VOP1 and VOP4 (mentionedin Materials and methods 2.3) were performed, according to the method described by Seabi et al. [18],by plating the C2C12 (mouse skeletal muscle, American Type Culture Collection [ATCC], Manassas,VA, USA) cells in 6-well plates at a density of 1 × 105 cells/mL. C2C12 cells, fully differentiated intomyotubules, were treated with 100 µL of 50 mg/µL, leaf extract for 3 h prior to the isolation of totalRNA. Insulin (10 µg/mL) was included as a control. Cells were detached from the culture platesand centrifuged (Beckman TJ-6, Analytical Instruments Brokers LLC, Golden Valley, MN, USA) at250× g for 5 min. Isolation of total RNA from C2C12 cells was carried out according to Seabi et al. [16],by centrifuging the harvested cells at 250× g for 5 min. Thereafter, RNA extraction was performedusing an RNA extraction kit (Life Technologies, Johannesburg, South Africa), and the RNA (0.5 µg) wasreverse transcribed to cDNA using the cDNA synthesis kit. The cDNA reaction mixture included 10 µLtemplate RNA, 2 µL oligo d (T) primer, 12 µL nuclease-free deionised water, 4 µL 5× reaction buffer,1 µL RibobLock RNase inhibitor and 1 µL MuLV reverse transcriptase [18]. The reaction was allowedat 42 ◦C for 60 min; subsequently the temperature was increased to 70 ◦C for 5 min to terminate thereaction. The polymerase chain reaction (PCR) was performed using a mixture of 2 µL of forward andreverse primers (0.4 µM each), 5 µL of template cDNA, 16 µL of nuclease-free sterile deionised waterand 25 µL of 2× ReadyMix (Kapa Biosystems, Wilmington, NC, USA) [18]. The conditions for the PCRreaction were similar to those reported by Seabi et al. [18]. After completion, the resulting products ofPCR were analysed on 2% agarose gel electrophoresis (Bio-Rad Laboratories, Sandton, Johannesburg,South Africa), at 75 V for at least 1 h at 25 ◦C, as described by Seabi et al. [18]. The primers used for thePCR (reverse and complementary) for GLUT 4 are given in Table S1.

2.6. Cell Cytotoxicity Using MTT Assay

Cell toxicity was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyltetrazoliumbromide, Merck, Johannesburg, South Africa) cytotoxicity assay using C2C12 myoblast cell (mouseskeletal muscle) line, according to a method described by Seabi, et al. [17] without any modifications.Cells were seeded at an initial cell density of 1 × 105 cells/mL in a 96-well cell culture plate.Thereafter, cells were treated with different concentrations (0.25–25 mg/mL) of the different cultivarsof cowpea leaf extracts (mentioned in Materials and methods 2.3) and incubated at 37 ◦C for 24 h.The untreated cells were included as the experimental control; ZnCl2 (0.25–2.5 mM) and H2O2(0.25–2.5%) were used as positive control. Afterwards, an aliquot of 20 µL of 5 mg/mL MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well and incubatedat 37 ◦C for an additional 4 h to allow the conversion of MTT to the coloured formazan. Cell cytotoxicity

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was measured at 570 nm using a microtitre-plate multimode detector (Promega-Glomax Multi-detectionsystem, Madison, WI, USA), using the formula below; the blank well included only the medium.

% Viable cells =∗abs sample− abs blank)dbs control− abs blank × 100

* abs—absorbance.

2.7. Carotenoids

Carotenoid extraction was performed according to Djuikwo, et al. [19], with some modifications.Powdered cowpea leaf (100 mg) was homogenised with acetone and 95% ethanol containing 0.1%(w/v) butylated hydroxytoluene (BHT) in an orbital shaker (RP1812, Paton Scientific, Victor Harbor, SA,Australia) for 10 min. The samples were saponified at 25 ◦C for 30 min in KOH (20% in methanol, w/v)while shaking at 100 rpm. Afterwards, hexane/dichloromethane mixture (70:30, v/v), containing 0.1%BHT, was added to extract carotenoid compounds into the upper phase. NaCl (10%, w/v) was addedfor phase separation, thereafter, centrifuged at 3900× g for 5 min at 25 ◦C (Eppendorf 5804, Lasec Pty,Midrand, South Africa). The upper layer was collected, combined and evaporated under nitrogenstream until dry. The crude extract was freshly reconstituted in methanol/MTBE (50:50, v/v), containing0.1% BHT, for UHPLC_UV_MSMS analysis.

Carotenoids were analysed using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific,Waltham, MA, USA) equipped with a Thermo UV detector, scanned at 450 nm, and a Thermo highresolution Q Exactive Quadrupole-Orbitrap mass spectrometer. Compound separation was performedon a YCM C30 column (3.6 × 250 mm, 3.6 µm) (Waters, Milford, MA, USA) maintained at 25 ◦C,with 0.1% formic acid in methanol (eluent A) and 0.1% formic acid in MTBE (eluent B). The gradientprogramme of mobile phase A was as follows: (0 min, 80%), (20 min, 75%), (30 min, 30%), (33 min,30%), (36 min, 80%), with the flow rate of 0.6 mL/min. Mass spectrometry analysis was operated inpositive mode, employing an atmospheric pressure chemical ionisation (APCI). A full MS scan (m/z120–1000) was acquired at a resolving power of 70,000 full-width half maximum. For the compoundsof interest, an MS/MS scan from m/z 80 to 650 was selected, with normalised collision energy at 20V.Carotenoids were quantified at 450 nm, using external calibration curves of carotenoid standardsstated in Section 2.1. Concentration of carotenoid standards was determined using a Cintra UV-Visspectrophotometer (GBC Scientific Equipment, Braeside, VIC, Australia), based on specific molarabsorption coefficients in solutions as described previously [19].

2.8. Amino Acids

Amino acids were quantified according to the method described by Mpai et al. [16]. Freeze-driedfrozen cowpea leaves (100 mg) were mixed with 6 N HCl and incubated in an oven at 110 ◦C for18 h; thereafter the mixture was cooled, centrifuged, filtered and dried in a speed vac concentrator.It was then derivative by adding 10 µL aliquot of the freshly made undiluted sample containing20 µL l-Norvaline in 80 µL of the sample to the 20 µL of AccQ-Tag Ultra amino acid kit, vortexedand incubated in the oven at 55 ◦C for 10 min. The vials were cooled for analysis using a WatersUPLC-PDA system (Waters, Milford, MA, USA). The conditions for UPLC analysis were similar to themethod described by Mpai et al. [16]; standard calibration curves were constructed to quantify theamino content and expressed as g/100 g.

2.9. Statistical Analysis

The experiments were repeated with two harvests within the season and the data adopted acompletely randomised design. As there was no significant variation between the two harvests,the data was pooled together for statistical analysis. For biological activities, three sample replicatesper leaf extract concentration per treatment (cowpea cultivars) were analysed, whereas for biochemicalanalysis a cumulative five replicate samples per treatment, (cowpea cultivars) were included. The data

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obtained were subjected to analysis of variance (ANOVA) using the statistical programme GenStatversion 11.1, statistical data analysis software (Hempstead, England, UK). Treatment means werecompared using Fishers protected t-test least. Significant difference (LSD) was at the 5% level ofsignificance. Pearson’s correlation coefficients were calculated to determine the strength of the linearrelationships between antioxidant capacity, targeted phenolic compounds and antidiabetic enzymeinhibition activity.

3. Results and Discussion

3.1. Quantification of Targeted Phenolic Metabolites in Cowpea Cultivars

Figure S1 illustrates the total ion chromatograms of phenolic metabolites from the leaves of cowpeacultivars operated in negative ESI-mode using a UPLC–QTOF/MS system. In total, seven compoundsthat belong to the group of phenolic acid and flavonoid glycosides were identified as main phenoliccompounds in cowpea cultivars including gentisic acid 5-O-glucoside, p-coumaric acid O-glucoside,ferulic acid O-glucosid and four quercetin derivatives (quercetin 3-sambubioside-3′-glucoside, quercetin3-glucosyl-(1->2)-galactoside, quercetin 3-(2G-xylosylrutinoside), and quercetin 3-O-rhamnoside7-O-glucosi (Table 1). The MS spectra of these compounds are given in Figure S2A–D.

Peak 1 (gentisic acid 5-O-glucoside) showed fragment ion at m/z 152 due to loss of hexoside [20].For peak 4 (quercetin 3-sambubioside-3′-glucoside), peak 5 (quercetin 3-glucosyl-(1->2)-galactoside)and peak 7 (quercetin 3-O-rhamnoside 7-O-glucoside) compounds had a fragment at m/z 301,which could be attributed to the release of quercetin (aglycone) [20–22] (Table 1 and Figure S1D,E,G).

Figure 2 presents the concentrations of seven phenolic metabolites in different cowpea cultivars.Concentration of gentisic acid 5-O-glucoside was significantly highest in cowpea cultivars VOP1(1087 mg/kg), compared to the other cowpea cultivars. The gentisic glucosides were reported in Bittermelon (Momordica charantia) [23], and Mutamba (Guazuma ulmifolia Lam) fruits [24].

Among the quercetin derivatives, quercetin 3-glucosyl-(1->2)-galactoside and quercetin3-(2G-xylosylrutinoside) were detected at higher concentrations compared to the other two quercetinderivatives. Quercetin 3-(2G-xylosylrutinoside) was the second dominant phenolic compound that isfound in green bean and the significantly highest concentration was found in cowpea cultivarVOP4 (653.4 mg/kg), followed by VOP1 (511.41 mg/kg) and VOP7 (489.47 mg/kg). Quercetin3-glucosyl-(1->2)-galactoside was obtained in cowpea cultivar VOP5 (653.4 mg/kg), followed byVOP2 (498.6 mg/kg) and VOP7 (486.2 mg/kg); other cowpea cultivars showed significantly lowerconcentrations of quercetin 3-glucosyl-(1->2)-galactoside. Cowpea cultivar VOP4 contained thehighest concentration of quercetin 3-sambubioside-3′-glucoside followed by VOP1 and VOP3. Highestconcentrations of quercetin 3-O-rhamnoside 7-O-glucoside was detected in cowpea cultivar VOP2.Concentrations of coumaric acid O-glucoside, and ferulic acid O-glucoside were highest in cowpeacultivars VOP4 and VOP8 respectively. Quercetin 3-O-xylosylrutinoside or isomers, were reportedpreviously in green beans [20–22].

Although the dietary phenolic acids and flavonoids were found in higher concentrations inthe cowpea cultivars VOP1 and VOP4, their health benefits depend on their bioavailability [25].Hollman et al. [26], reported the bioavailability of quercetin glycosides in onions and the pure quercetinrutinoside at 52% and 17%, respectively. Ferulic acid has showed efficient absorption when it exits asfree from in tomatoes or beers, but its bioavailability is limited in the ester forms reported in cereals [27].Further studies on digestive stability, bio accessibility, bioavailability and subsequently bioactivity,both in vitro and in vivo, are strongly recommended to get a better understanding of nutritional valuesof cowpea leaves, an emerging food in the African market. Heat map (Figure 2) demonstrated thequantitative pattern of phenolic metabolites in the leaves of different cowpea cultivars. The patternand magnitude, relating to the colour intensity (hue) from +2 to −2, with 0 as symmetry, relate tovisualisation of response intensities of 19 compounds, including the unidentified compounds.

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Table 1. Identification of phenolic compounds in different cowpea cultivars by a UPLC–QTOF/MS data.

Peak Retention Time M-H M-H Formula ppm Error MSE Fragments UV Identification

1 3.29 315.0697 C13H15O9 −5.1 152,108 152,108 Gentisic acid 5-O-glucoside2 4.51 325.0889 C15H17O8 −1.2 163,145,119 289 Coumaric acid O-glucoside3 4.84 355.1024 C16H19O9 −1.4 261,243,193,175,160,134 321 Ferulic acid O-glucoside4 5.33 757.1812 C57H25O3 1.1 301,271,197 255,351 Quercetin 3-sambubioside-3′-glucoside5 5.60 625.1356 C34H25O12 1.6 301,284,271,255,178,155 255 Quercetin 3-glucosyl-(1->2)-galactoside6 5.70 741.1879 C32H37O20 0.1 625,443,355,285 265,335 Quercetin 3-(2G-xylosylrutinoside)7 5.95 609.1493 C27H29O16 3.8 595,361,301,271,255 257,333 Quercetin 3-O-rhamnoside 7-O-glucoside

Foods 2020, 9, 1285 9 of 23


Figure 2. Concentration of targeted phenolic compounds in different cowpea cultivars. FW—fresh weight, Bar with same alphabets are not significantly different between cultivars at p < 0.05 for a specific phenolic compound. Data obtained were subjected to analysis of variance (ANOVA) using the statistical programme. Each bar represents the mean and standard deviation (n = 3). Gentisic acid 5-O-glucoside (G-A-O-g); p-Coumaric acid O-glucoside (CA-O-g); Ferulic acid O-glucoside (FA-O-g); Quercetin 3-sambubioside-3′-glucoside (Q-S-g); Quercetin 3-glucosyl-(1->2)-galactoside (Q-G-g); Quercetin 3-(2G-xylosylrutinoside) (Q-G-X-r); Quercetin 3-O-rhamnoside 7-O-glucoside (Q-r-g).

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Figure 2. Concentration of targeted phenolic compounds in different cowpea cultivars. FW—fresh weight, Bar with same alphabets are not significantly differentbetween cultivars at p < 0.05 for a specific phenolic compound. Data obtained were subjected to analysis of variance (ANOVA) using the statistical programme.Each bar represents the mean and standard deviation (n = 3). Gentisic acid 5-O-glucoside (G-A-O-g); p-Coumaric acid O-glucoside (CA-O-g); Ferulic acid O-glucoside(FA-O-g); Quercetin 3-sambubioside-3′-glucoside (Q-S-g); Quercetin 3-glucosyl-(1->2)-galactoside (Q-G-g); Quercetin 3-(2G-xylosylrutinoside) (Q-G-X-r); Quercetin3-O-rhamnoside 7-O-glucoside (Q-r-g).

Foods 2020, 9, 1285 10 of 23

3.2. Multivariate Analysis

The unsupervised PCA illustrates clustering different cowpea cultivars (Figure S3), VOP1 cluster,the bigger cluster (VOP2-7) and VOP8 cluster. The PC 1 and PC 2, described for more than 70% ofthe variance, separated the cowpea cultivar VOP1 and VOP8 from the rest of the cultivars alongthe PC1 and PC2, respectively. A supervised orthogonal projection to latent structure-discriminantanalysis (OPLS) model was performed in order to understand the separation of the clustered groups ofcowpea cultivars, clearly based on their phenolic metabolites. In the S-plot (Figure 3), the compoundsfurther along the x-axis contributed substantially to the variance between the groups, whilst thefurther the Y-axis, the higher the accuracy of the analytical result [15]. Therefore, in the S-plot, anunidentified compound ([M − H]− 127.0020, m/z 2.82), presented in the upper right quadrant, showedthe higher concentrations in cowpea cultivars VOP2, VOP3, VOP4, VOP5 VOP7 and VOP8 (Figure 4),whilst gentisicacid 5-O-glucoside, located at the lower left quadrant, showed higher concentration incowpea cultivar VOP1. Thus, gentisicacid 5-O-glucoside is the marker candidate for the separationof VOP1 from the bigger cluster (VOP2-7) and the VOP8 which are not district from each other.Furthermore, the quantitative difference of the unidentified compound ([M − H]− 127.0020) andgentisic acid 5-O-glucoside (eluted at Rt 2.82 and 3.27) revealed abundance at 50 and 200 peak intensityin counts/s respectively, in cowpea cultivar VOP1 (Figure S4). Samples from other cowpea cultivars,VOP2, VOP3, VOP4, VOP5, VOP7 and VOP8, demonstrated at Rt 2.82 and 3.27 the abundance of bothunidentified compound ([M −H]− 127.0020) and gentisic acid 5-O-glucoside at 50 peak intensity incounts/s (Figure S4).


Figure 3. Score plot of orthogonal partial least squares discriminant analysis of UPLC–Q-TOF/MS spectra of the leaves of different cowpea cultivars. Gentisic acid 5-O-glucoside (G-A-O-g), Un—Unidentified compound.

G-A-O-g

Un

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prof

ile p

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r [1]

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Covariance P1

Figure 3. Score plot of orthogonal partial least squares discriminant analysis of UPLC–Q-TOF/MS spectra of the leaves of different cowpea cultivars. Gentisic acid5-O-glucoside (G-A-O-g), Un—Unidentified compound.

Foods 2020, 9, 1285 12 of 23


Figure 4. Heat map of nineteen phenolic metabolites (variables) in hierarchical clustering in the leaves of cowpea cultivars. Gentisic acid 5-O-glucoside (G-A-O-g); p-Coumaric acid O-glucoside (CA-O-g); Ferulic acid O-glucoside (FA-O-g); Quercetin 3-sambubioside-3′-glucoside (Q-S-g); Quercetin 3-glucosyl-(1->2)-galactoside (Q-G-g); Quercetin 3-(2G-xylosylrutinoside) (Q-G-X-r); Quercetin 3-O-rhamnoside 7-O-glucoside (Q-r-g). The pattern and magnitude relating to the colour intensity (hue) from +2 to −2 and 0 as symmetry) relating to visualization of response intensities of 19 compounds (identified and unidentified compounds) present in theses cowpea cultivars.

Figure 4. Heat map of nineteen phenolic metabolites (variables) in hierarchical clustering in the leaves of cowpea cultivars. Gentisic acid 5-O-glucoside (G-A-O-g);p-Coumaric acid O-glucoside (CA-O-g); Ferulic acid O-glucoside (FA-O-g); Quercetin 3-sambubioside-3′-glucoside (Q-S-g); Quercetin 3-glucosyl-(1->2)-galactoside(Q-G-g); Quercetin 3-(2G-xylosylrutinoside) (Q-G-X-r); Quercetin 3-O-rhamnoside 7-O-glucoside (Q-r-g). The pattern and magnitude relating to the colour intensity(hue) from +2 to −2 and 0 as symmetry) relating to visualization of response intensities of 19 compounds (identified and unidentified compounds) present in thesescowpea cultivars.

Foods 2020, 9, 1285 13 of 23

3.3. In Vitro Antioxidant Capacity

FRAP assay was selected in this study because it is a quick and simple method to conduct, providesreproducible results and readily relates to the molar concentration of the antioxidants available incowpea leaves. Results from in vitro antioxidant capacity (FRAP assay) are shown in Figure 5.Antioxidant capacity varied among the cultivars studied and VOP1 exhibited the strongest antioxidantcapacity followed by VOP4. The FRAP activity in the leaf extracts of cowpea cultivar VOP1 is higherthan that in indigenous fruits and vegetables, such as tree tomato (Cyphomandra betacea) at ripe stage(1.62 mmol TEC/100 g), and spider plant (Cleome gynandra L.; 1.56 mmol TEC/100 g) [28]. Indigenousvegetable amaranth leaves (Amaranthus spinosus; 1 mmol TEC/100 g) and commercial vegetable spinach,unknown cultivar (0.98 mmol TEC/100 g) [26], which showed lower FRAP activity than the leavesof cowpea cultivars VOP1, VOP4 and VOP8. Similarly, sweet potato leaves (Solanum macrocarpon L;0.87 mmol TEC/100 g) [28] showed lower FRAP activity than the cowpea cultivars VOP1, VOP4, VOP8,VOP7 and VOP5.


3.3. In Vitro Antioxidant Capacity

FRAP assay was selected in this study because it is a quick and simple method to conduct, provides reproducible results and readily relates to the molar concentration of the antioxidants available in cowpea leaves. Results from in vitro antioxidant capacity (FRAP assay) are shown in Figure 5. Antioxidant capacity varied among the cultivars studied and VOP1 exhibited the strongest antioxidant capacity followed by VOP4. The FRAP activity in the leaf extracts of cowpea cultivar VOP1 is higher than that in indigenous fruits and vegetables, such as tree tomato (Cyphomandra betacea) at ripe stage (1.62 mmol TEC/100 g), and spider plant (Cleome gynandra L.; 1.56 mmol TEC/100 g) [28]. Indigenous vegetable amaranth leaves (Amaranthus spinosus; 1 mmol TEC/100 g) and commercial vegetable spinach, unknown cultivar (0.98 mmol TEC/100 g) [26], which showed lower FRAP activity than the leaves of cowpea cultivars VOP1, VOP4 and VOP8. Similarly, sweet potato leaves (Solanum macrocarpon L; 0.87 mmol TEC/100 g) [28] showed lower FRAP activity than the cowpea cultivars VOP1, VOP4, VOP8, VOP7 and VOP5.

Amongst the commercial fruits, banana (1.4 mmol TEC/100 g) and orange (1.2 mmol TEC/100 g) [28] demonstrated lower FRAP activity than the cowpea cultivars VOP1 and VOP4. Commercial fruit, papaya (0.89 mmol TEC/100 g) [28], showed a relatively similar level of FRAP activity as cowpea cultivars VOP2 and VOP7, but lower than VOP1, VOP4, VOP8, and VOP3. FRAP activity of passion fruit (7.2 mmol TEC/100 g) [28] similarly coincided with the activity of cowpea cultivar VOP5, however, all the leaves of other cowpea cultivars showed higher FRAP activity. Amongst the vegetables, brown beans (Phaseolus vulgaris L; 7.10 mmol TEC/100 g), sweet pepper (0.38 mmol TEC/100 g), tomato (0.38 mmol TEC/100 g), French beans (Phaseolus vulgaris L.; 0.21 mmol TEC/100 g), and sweet potato (Ipomoea batatas L. Lam.; 0.15 mmol TEC/100 g) [28] showed lower FRAP activity when compared to the leaves of all cowpea cultivars. In addition, cowpea cultivars VOP1 to VOP8 showed lower FRAP activity compared to seeds the seeds of Faba bean (Vicia faba) accessions that varied from 56.3 to 103.5 mmol TEC/100 g [29].

Figure 5. Antioxidant capacity of the leaves of different cowpea (leaves) cultivars. Bar with same alphabets are not significantly different between cultivars at p < 0.05. Data obtained were subjected to analysis of variance (ANOVA) using the statistical programme. Each bar represents the mean and standard deviation (n = 5).

3.4. In Vitro Cytotoxic Effect

The cytotoxic effects of leaf extracts of cowpea cultivars on C2C12 myoblast cell line are given in percentage cell viability shown in Figure S5. All seven cowpea cultivars tested, using the C2C12 muscle cells, demonstrated absence of inhibitions on cell viability at 50% for the three concentrations 0.25, 0.5

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Figure 5. Antioxidant capacity of the leaves of different cowpea (leaves) cultivars. Bar with samealphabets are not significantly different between cultivars at p < 0.05. Data obtained were subjectedto analysis of variance (ANOVA) using the statistical programme. Each bar represents the mean andstandard deviation (n = 5).

Amongst the commercial fruits, banana (1.4 mmol TEC/100 g) and orange (1.2 mmol TEC/100 g) [28]demonstrated lower FRAP activity than the cowpea cultivars VOP1 and VOP4. Commercial fruit,papaya (0.89 mmol TEC/100 g) [28], showed a relatively similar level of FRAP activity as cowpeacultivars VOP2 and VOP7, but lower than VOP1, VOP4, VOP8, and VOP3. FRAP activity of passionfruit (7.2 mmol TEC/100 g) [28] similarly coincided with the activity of cowpea cultivar VOP5, however,all the leaves of other cowpea cultivars showed higher FRAP activity. Amongst the vegetables, brownbeans (Phaseolus vulgaris L; 7.10 mmol TEC/100 g), sweet pepper (0.38 mmol TEC/100 g), tomato(0.38 mmol TEC/100 g), French beans (Phaseolus vulgaris L.; 0.21 mmol TEC/100 g), and sweet potato(Ipomoea batatas L. Lam.; 0.15 mmol TEC/100 g) [28] showed lower FRAP activity when comparedto the leaves of all cowpea cultivars. In addition, cowpea cultivars VOP1 to VOP8 showed lowerFRAP activity compared to seeds the seeds of Faba bean (Vicia faba) accessions that varied from 56.3 to103.5 mmol TEC/100 g [29].

3.4. In Vitro Cytotoxic Effect

The cytotoxic effects of leaf extracts of cowpea cultivars on C2C12 myoblast cell line are givenin percentage cell viability shown in Figure S5. All seven cowpea cultivars tested, using the C2C12muscle cells, demonstrated absence of inhibitions on cell viability at 50% for the three concentrations

Foods 2020, 9, 1285 14 of 23

0.25, 0.5 and 1 mg/mL after 24 h incubation, whilst the highest toxicity was exhibited by the control(H2O2). Thus, all cowpea cultivars tested using this assay did not exhibit a strong enough toxicity toC2C12 myoblast cell lines at all tested concentrations.

3.5. Antidiabetic Effects and GLUT4 mRNA Levels

Figure 6 illustrates the percentage inhibition of α-glucosidase of the leaf extracts of differentcowpea cultivars at concentrations 6.25, 25 and 50 mg/mL using glucose as the substrate. The leafextract of cowpea cultivar VOP1 at 25 and 50 mg/mL demonstrated the significantly higher percentageinhibitory values (86% and 93%) than the commercial inhibitor (Acarbose), whilst the leaf extract ofVOP1 at 6.25 mg/mL revealed an almost similar percentage of inhibition (76%) as the commercialinhibitor (at 5 mg/mL). The cowpea leaf extracts of VOP4 at 50 mg/mL showed similar percentageof inhibition (80%) as the commercial inhibitor. The percentage inhibitory value of α-glucosidasein cowpea cultivar VOP4 was significantly lower than the VOP1 at all three concentrations tested;however, cowpea leaf extracts of the cultivar VOP4 showed higher inhibitory activity compared tocultivars VOP2, VOP3, VOP5, VOP7 and VOP8 at all the three tested levels.


and 1 mg/mL after 24 h incubation, whilst the highest toxicity was exhibited by the control (H2O2). Thus, all cowpea cultivars tested using this assay did not exhibit a strong enough toxicity to C2C12 myoblast cell lines at all tested concentrations.

3.5. Antidiabetic Effects and GLUT4 mRNA Levels

Figure 6 illustrates the percentage inhibition of α-glucosidase of the leaf extracts of different cowpea cultivars at concentrations 6.25, 25 and 50 mg/mL using glucose as the substrate. The leaf extract of cowpea cultivar VOP1 at 25 and 50 mg/mL demonstrated the significantly higher percentage inhibitory values (86% and 93%) than the commercial inhibitor (Acarbose), whilst the leaf extract of VOP1 at 6.25 mg/mL revealed an almost similar percentage of inhibition (76%) as the commercial inhibitor (at 5 mg/mL). The cowpea leaf extracts of VOP4 at 50 mg/mL showed similar percentage of inhibition (80%) as the commercial inhibitor. The percentage inhibitory value of α-glucosidase in cowpea cultivar VOP4 was significantly lower than the VOP1 at all three concentrations tested; however, cowpea leaf extracts of the cultivar VOP4 showed higher inhibitory activity compared to cultivars VOP2, VOP3, VOP5, VOP7 and VOP8 at all the three tested levels.

The inhibition of α-amylase activity is shown in Figure 7. The leaf extracts of cowpea cultivars VOP1 and VOP4, at the concentration of 50 mg/mL, possessed the highest inhibitory activity (91–94%) compared to the commercial inhibitor (Acarbose), at the same level. A similar trend in results was also found for leaf extracts of cowpea accessions VOP1 and VOP4 at the intermediate concentration of 25 mg/mL, where they showed significantly higher inhibitory effects (75% and 76%) compared to the activity of the commercial inhibitor; however, leaf extracts of VOP1 and VOP4 at the lowest concentration of 6.25 mg/mL revealed a similar inhibitory effect (58%) as the commercial inhibitor. Overall, leaf extracts of cowpea cultivars VOP1 and VOP4 demonstrated the highest inhibitory effect on α-amylase and α-glucosidase activity among the cultivars studied.

Figure 6. Percentage inhibition of leaf extracts of cowpea cultivars against α-glucosidase. Bars representing the enzyme activity for a specific leaf extract concentration with Bar with same alphabets are not significantly different between cultivars at p < 0.05. Data obtained were subjected to analysis of variance (ANOVA) using the statistical programme. Each bar represents the mean and standard deviation (n = 3).

Figure 6. Percentage inhibition of leaf extracts of cowpea cultivars against α-glucosidase.Bars representing the enzyme activity for a specific leaf extract concentration with Bar with samealphabets are not significantly different between cultivars at p < 0.05. Data obtained were subjectedto analysis of variance (ANOVA) using the statistical programme. Each bar represents the mean andstandard deviation (n = 3).

The inhibition of α-amylase activity is shown in Figure 7. The leaf extracts of cowpea cultivarsVOP1 and VOP4, at the concentration of 50 mg/mL, possessed the highest inhibitory activity (91–94%)compared to the commercial inhibitor (Acarbose), at the same level. A similar trend in results wasalso found for leaf extracts of cowpea accessions VOP1 and VOP4 at the intermediate concentrationof 25 mg/mL, where they showed significantly higher inhibitory effects (75% and 76%) comparedto the activity of the commercial inhibitor; however, leaf extracts of VOP1 and VOP4 at the lowestconcentration of 6.25 mg/mL revealed a similar inhibitory effect (58%) as the commercial inhibitor.Overall, leaf extracts of cowpea cultivars VOP1 and VOP4 demonstrated the highest inhibitory effecton α-amylase and α-glucosidase activity among the cultivars studied.


Figure 7. Percentage inhibition of leaf extracts of cowpea cultivars against α-amylase activities. Bars representing the enzyme activity for a specific leaf extract concentration. Bar with same alphabets are not significantly different between cultivars at p < 0.05. Data obtained were subjected to analysis of variance (ANOVA) using the statistical programme. Each bar represents the mean and standard deviation (n = 3).

Inhibition of enzymes, such as α-glucosidase and α-amylase, which are associated with carbohydrate digestion, is an important approach to reduce the postprandial hyperglycaemia [6]. Leaf extracts (at 50 mg/mL) of cowpea cultivar VOP1 revealed higher inhibitory activity of α-glucosidase and α-amylase compared to the leaf extracts of Moringa leaves (dried) [30]. Furthermore, cowpea cultivar VOP1 (50 µL/mL) showed more or less similar α-amylase inhibitory activity as blueberry cultivars, Blueray and Blur crop, grown in Southern Illinois, USA [31].

Rasouli et al. [32] explained that the presence of OH groups in positions 3 (ring C), 7 (ring A), 4 and 5 (ring B) in polyphenol molecular structure play a vital role in the inhibitory effects of the α-glucosidase and α-amylase activities. In addition, the total number of hydroxyl groups, C-2-C-3 double bond, and C-4 ketonic functional group play a major role in anti-diabetic effect. Furthermore, coumaric acid glycosides demonstrated greater inhibitory activities on these enzymes than the free (non-glyosidic) p-coumaric acid [33]. The observed difference in the degree of inhibition of these two enzymes could be due to the synergistic effect of different phenolic compounds and their varying concentrations [34].

The influence of polyphenols of VOP1 and VOP4 on expression levels of GLUT4 and GAPDH genes in C2C12 cells is shown in Figure 6. Leaf extracts of cowpea cultivars VOP4 significantly upregulated the GLUT4 gene to a similar level as the comparative control treatment (insulin) (Figure 8). This result indicated higher glucose uptake by the C2C12 cells activated by the pool of phenolic compounds present in leaf extracts of cowpea cultivars VOP1 and VOP4. Boue et al. [35] demonstrated the influence of phenolic compounds on GLUT4 mRNA levels in two pigmented rice bran extracts and stated the positive effects on long-term regulation of glucose transport.

Tea flavonol glycosides, which predominantly include quercetin 3-O-glucosyl-rhamnosyl-glucoside, showed significant differences with regard to glucose homeostasis in a type 2 diabetes mouse model after administration of flavonol-rich tea cultivars [36]. Ferulic acid, containing p-hydroxy and m-methoxy structures, was reported as one of the compounds that effectively enhanced insulin secretion [33]. This study indicates that the different phenolic compounds in cowpea leaves are responsible for the observed anti-diabetic activity, and this activity depends on the concentration of cowpea (VOP1 and VOP4) leaf extracts, and the specific molecular structure of the phenolic compounds.

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Figure 7. Percentage inhibition of leaf extracts of cowpea cultivars against α-amylase activities.Bars representing the enzyme activity for a specific leaf extract concentration. Bar with same alphabetsare not significantly different between cultivars at p < 0.05. Data obtained were subjected to analysisof variance (ANOVA) using the statistical programme. Each bar represents the mean and standarddeviation (n = 3).

Inhibition of enzymes, such as α-glucosidase and α-amylase, which are associated withcarbohydrate digestion, is an important approach to reduce the postprandial hyperglycaemia [6].Leaf extracts (at 50 mg/mL) of cowpea cultivar VOP1 revealed higher inhibitory activity ofα-glucosidaseand α-amylase compared to the leaf extracts of Moringa leaves (dried) [30]. Furthermore, cowpeacultivar VOP1 (50 µL/mL) showed more or less similar α-amylase inhibitory activity as blueberrycultivars, Blueray and Blur crop, grown in Southern Illinois, USA [31].

Rasouli et al. [32] explained that the presence of OH groups in positions 3 (ring C), 7 (ring A),4 and 5 (ring B) in polyphenol molecular structure play a vital role in the inhibitory effects of theα-glucosidase and α-amylase activities. In addition, the total number of hydroxyl groups, C-2-C-3double bond, and C-4 ketonic functional group play a major role in anti-diabetic effect. Furthermore,coumaric acid glycosides demonstrated greater inhibitory activities on these enzymes than the free(non-glyosidic) p-coumaric acid [33]. The observed difference in the degree of inhibition of these twoenzymes could be due to the synergistic effect of different phenolic compounds and their varyingconcentrations [34].

The influence of polyphenols of VOP1 and VOP4 on expression levels of GLUT4 and GAPDHgenes in C2C12 cells is shown in Figure 6. Leaf extracts of cowpea cultivars VOP4 significantlyupregulated the GLUT4 gene to a similar level as the comparative control treatment (insulin) (Figure 8).This result indicated higher glucose uptake by the C2C12 cells activated by the pool of phenoliccompounds present in leaf extracts of cowpea cultivars VOP1 and VOP4. Boue et al. [35] demonstratedthe influence of phenolic compounds on GLUT4 mRNA levels in two pigmented rice bran extracts andstated the positive effects on long-term regulation of glucose transport.


Figure 8. The GLUT4 gene expression in C2C12 skeletal mouse muscle cells in presence of leaf extracts 50 mg/µL (100 µL) of cowpea cultivars VOP1 and VOP4 after 3 h of incubation. NC—negative control, PC—positive control (Insulin). Bar with same alphabets are not significantly different between cultivars at p < 0.05. Data obtained were subjected to analysis of variance (ANOVA) using the statistical programme. Each bar represents the mean and standard deviation (n = 3).

3.6. Pearson’s Correlation Analysis

There were positive correlations between the antioxidant capacity (FRAP activity) and gentisic acid-5-O-glucoside, coumaric acid O-glucoside, ferulic acid O-glucoside, quercetin 3-glucosyl-(1->2)-galactoside, quercetin 3-sambubioside-3′-glucoside, Quercetin 3-(2G-xylosylrutinoside and quercetin 3-O-rhamnoside 7-O-glucoside (Table S2). Similarly, phenolic components, gentisic acid 5-O-glucoside, coumaric acid O-glucoside, ferulic acid O-glucoside, quercetin 3-glucosyl-(1->2)-galactoside, quercetin 3-sambubioside-3′-glucoside, quercetin 3-(2G-xylosylrutinoside) and quercetin 3-O-rhamnoside 7-O-glucoside from cowpea leaf extract revealed a positive correlation with α-glucosidase and α-amylase activity (Table S2). In addition, significantly positive correlations between FRAP activity and both α-glucosidase (R2 = 0.73, p < 0.05) and α-amylase (R2 = 0.80, p < 0.05) inhibition were also observed from the results of Pearson’s correlation analysis. It is evident from this study that the observed differences in antioxidant capacity between different cowpea cultivars could be related to the different concentrations of phenolic compounds. Although, antioxidant capacities and the concentrations of different phenolic compounds are affected by the geographical locations and altitude difference [37], in this case the plants were grown under the same environment and the observed differences in the concentrations of phenolic compounds and the antioxidant capacities are probably due to the genetic makeup of the cultivars.

3.7. Carotenoid Profile in Cowpea Cultivars

In general, cowpea cultivars VOP3 demonstrated significantly high total carotenoids content (Table 2), with lutein and β-carotene mainly contributing to the total carotenoid content. Table S3 illustrates the characterisation of carotenoid compounds detected in cowpea accessions by UHPLC–APCI-MS analysis. Figure S6A,B and Table S2 demonstrate the identification of the detected carotenoid components. Lutein concentration showed the following trend in cowpea cultivars: VOP2 > VOP8 > VOP3 > VOP1 > VOP4 > VOP7 > VOP5 (Table 3). Corn (0.092 mg/100 g), onion stalk (0.923 mg/100 g), broccoli (0.616 mg/100 g), capsicum (0.367 mg/100 g) [38], black Nightshade leaves (Solanum nigrum) (84.86 mg/100 g) [39] and carrot (42.0 mg/100 g) [40] had lower concentrations of lutein compared to the concentrations detected in cowpea cultivars. Zeaxanthin, was found at a minor proportion in cowpea cultivars, only ranged from 0.04 to 0.09 mg/100 g, which was lower than that reported in corn (0.28 mg/100 g) and onion stalk (0.305 mg/100 g) [41], but comparable to broccoli (0.04 mg/100 g)

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Figure 8. The GLUT4 gene expression in C2C12 skeletal mouse muscle cells in presence of leaf extracts50 mg/µL (100 µL) of cowpea cultivars VOP1 and VOP4 after 3 h of incubation. NC—negative control,PC—positive control (Insulin). Bar with same alphabets are not significantly different between cultivarsat p < 0.05. Data obtained were subjected to analysis of variance (ANOVA) using the statisticalprogramme. Each bar represents the mean and standard deviation (n = 3).

Tea flavonol glycosides, which predominantly include quercetin 3-O-glucosyl-rhamnosyl-glucoside, showed significant differences with regard to glucose homeostasis in a type 2 diabetesmouse model after administration of flavonol-rich tea cultivars [36]. Ferulic acid, containing p-hydroxyand m-methoxy structures, was reported as one of the compounds that effectively enhanced insulinsecretion [33]. This study indicates that the different phenolic compounds in cowpea leaves areresponsible for the observed anti-diabetic activity, and this activity depends on the concentration ofcowpea (VOP1 and VOP4) leaf extracts, and the specific molecular structure of the phenolic compounds.

3.6. Pearson’s Correlation Analysis

There were positive correlations between the antioxidant capacity (FRAP activity)and gentisic acid-5-O-glucoside, coumaric acid O-glucoside, ferulic acid O-glucoside,quercetin 3-glucosyl-(1->2)-galactoside, quercetin 3-sambubioside-3′-glucoside, Quercetin3-(2G-xylosylrutinoside and quercetin 3-O-rhamnoside 7-O-glucoside (Table S2). Similarly,phenolic components, gentisic acid 5-O-glucoside, coumaric acid O-glucoside, ferulic acidO-glucoside, quercetin 3-glucosyl-(1->2)-galactoside, quercetin 3-sambubioside-3′-glucoside, quercetin3-(2G-xylosylrutinoside) and quercetin 3-O-rhamnoside 7-O-glucoside from cowpea leaf extractrevealed a positive correlation with α-glucosidase and α-amylase activity (Table S2). In addition,significantly positive correlations between FRAP activity and both α-glucosidase (R2 = 0.73, p < 0.05)and α-amylase (R2 = 0.80, p < 0.05) inhibition were also observed from the results of Pearson’scorrelation analysis. It is evident from this study that the observed differences in antioxidant capacitybetween different cowpea cultivars could be related to the different concentrations of phenoliccompounds. Although, antioxidant capacities and the concentrations of different phenolic compoundsare affected by the geographical locations and altitude difference [37], in this case the plants weregrown under the same environment and the observed differences in the concentrations of phenoliccompounds and the antioxidant capacities are probably due to the genetic makeup of the cultivars.

3.7. Carotenoid Profile in Cowpea Cultivars

In general, cowpea cultivars VOP3 demonstrated significantly high total carotenoids content(Table 2), with lutein and β-carotene mainly contributing to the total carotenoid content. Table S3illustrates the characterisation of carotenoid compounds detected in cowpea accessions byUHPLC–APCI-MS analysis. Figure S6A,B and Table S2 demonstrate the identification of the detectedcarotenoid components. Lutein concentration showed the following trend in cowpea cultivars:

Foods 2020, 9, 1285 17 of 23

VOP2 > VOP8 > VOP3 > VOP1 > VOP4 > VOP7 > VOP5 (Table 3). Corn (0.092 mg/100 g), onion stalk(0.923 mg/100 g), broccoli (0.616 mg/100 g), capsicum (0.367 mg/100 g) [38], black Nightshade leaves(Solanum nigrum) (84.86 mg/100 g) [39] and carrot (42.0 mg/100 g) [40] had lower concentrations oflutein compared to the concentrations detected in cowpea cultivars. Zeaxanthin, was found at a minorproportion in cowpea cultivars, only ranged from 0.04 to 0.09 mg/100 g, which was lower than thatreported in corn (0.28 mg/100 g) and onion stalk (0.305 mg/100 g) [41], but comparable to broccoli(0.04 mg/100 g) [41].Violaxanthin content was highest in VOP3 (24.9 mg/100 g), whilst the VOP5showed the lowest violaxanthin content (8.4 mg/100 g) (Table 2). Onion stalks (1.83 mg/100 g), beetrootleaves (3.97 mg/100 g), carrot greens (7.00 mg/100 g) and broccoli (1.45 mg/100 g) [39] demonstratedlower violaxanthin content compared to the leaves of all cowpea cultivars. Conversely, corianderleaves (83.43 mg/100 g), amaranthus (Amaranthus viridis) (84.06 mg/100 g) [39], on dry weight basis,showed higher violaxanthin concentrations compared to the leaves of the studied cowpea cultivars.However, black Nightshade leaves (Solanum nigrum) (22.17 mg/100 g) [39] showed more or less similarconcentrations of violoxanthin as VO3. Significantly, highest concentrations of all-trans-β-carotenewere detected in cowpea cultivars VOP1, whereas VO5 showed the lowest concentrations (Table 2).Additionally, a trace amount of 9-cis-beta-carotene (2.4–3.6 mg/100 g) was also found in all cowpeacultivars (Table 2).

The results indicate that lutein and beta-carotene are predominant carotenoids in cowpea leaves,which are beneficial to human health. For instance, lutein and zeaxanthin are well known as importantcomponents of the human macula and retina [42]. Increased intake of lutein proved to correlatepositively with increased macular pigment density, reducing the risk of macular degeneration byproviding antioxidant protection against the damaging blue light [42]. The recommended daily levelsfor eye health are 10 mg/day of lutein and 2 mg/day of zeaxanthin for adults [43]. Thus, a serving ofapproximately 10 g of cowpea leaf powder added to a soup (except for VOP5) will fulfil the dailyrecommendation of lutein required for adults. However, it should be noted that the bioavailabilityof lutein depends on food preparation and cooking methods, therefore, follow up studies need to beinvestigated using both in vitro digestion models and human clinical trials to confirm the health-relatedbenefits. Therefore, the cowpea cultivar VOP2 is promising for food supplementation programmes toreduce the risk of age-related macular degeneration.

Violaxanthin demonstrated higher antioxidant, anti-inflammatory and anti-proliferativeactivities [44]. All-trans-β-carotene is the predominant isomer in many fresh fruits and vegetables.The ratio of 9-cis to all-trans-β-carotene in papaya was higher (0.66) compared to the cowpea cultivars,VOP1 (0.034), VOP3 (0.042) and VOP8 (0.03). Furthermore, the cis-isomers were reported to increaseduring food processing, cooking methods and digestive metabolism in the intestine [45]. Previousreports based on different models suggest that the cis-isomers are preferred to trans-isomers andpossess higher antioxidant potency [46].

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Table 2. Carotenoid content in the leaves of different cowpea cultivars.

Cowpea Cultivars Violaxanthin Lutein Zeaxanthin All-Trans-Beta-Carotene 9-Cis-Beta-Carotene Total Carotenoids

mg/100 g DW

VPO1 17.8 ± 1.5 *,c 109.1 ± 8.6d 0.04 ± 0.01d 92.6 ± 1.7a 3.2 ± 0.2b 222.7 ± 1.0bVPO2 20.4 ± 0.7b 124.6 ± 1.8a 0.06 ± 0.02c 71.1 ± 1.7d 3.0 ± 2.6c 220.7 ± 1.6cVPO3 24.9 ± 6.0a 111.2 ± 1.7c 0.04 ± 0.01d 84.7 ± 5.9c 3.6 ± 0.3a 224.5 ± 1.0aVPO4 16.9 ± 0.7d 99.9 ± 9.5e 0.06 ± 0.01c 59.3 ± 8.7e 3.3 ± 0.2b 179.6 ± 1.5eVPO5 8.4 ± 1.3f 74.5 ± 1.0g 0.10 ± 0.03a 43.0 ± 4.9f 2.4 ± 0.1e 129.7 ± 1.5fVPO7 15.2 ± 1.9e 94.3 ± 1.0f 0.04 ± 0.04d 70.3 ± 1.3d 2.7 ± 0.1d 181.8 ± 1.0dVPO8 15.5 ± 1.1e 116.5 ± 1.2b 0.09 ± 0.01b 87.2 ± 5.2b 2.9 ± 0.1d 222.2 ± 1.5b

DW—dry weight, * Data present mean and standard deviation. Different letters at the same column indicate significant differences at (p < 0.05).

Table 3. Percentage of total protein, non-essential, essential and branched amino acids in different cowpea cultivars.

Cowpea Cultivars Total Protein (g/100 g DW) % Non-Essential Amino Acids % Essential Amino Acids % Branched-Chain Amino Acids

VOP1 28.4 ± 0.1 *,d 63.61 ± 0.2a 36.7 ± 0.2c 17.0 ± 0.2dVOP2 25.1 ± 0.1f 59.20 ± 0.1d 40.8 ± 0.1a 19.8 ± 0.0aVOP3 30.2 ± 0.2b 59.40 ± 0.1d 40.6 ± 0.1a 18.4 ± 0.3bcVOP4 31.3 ± 0.3a 61.30 ± 0.7c 38.7 ± 0.7b 18.1 ± 0.6cVOP5 29.3 ± 0.2c 62.50 ± 0.3b 37.5 ± 0.3c 18.6 ± 0.3bcVOP7 28.3 ± 0.2d 59.50 ± 0.2d 40.5 ± 0.2a 19.2 ± 0.1abVOP8 27.2 ± 0.3e 61.50 ± 0.3bc 38.5 ± 0.3b 17.9 ± 0.2b

* Data present mean and standard deviation (n = 3). Different letters at the same column indicate significant differences at (p < 0.05).

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3.8. Amino Acid Components in Cowpea Cultivars

The ratios of essential amino acids and branched amino acids to the total protein content werein the ranges of 36.7–40.8% and 17–19.8%, respectively (Table 3). The highest total protein contentwas detected in cowpea cultivars VOP3 (30.2 g/100 g) and VOP4 (31.3 g/100 g) compared to the othercultivars. Cowpea cultivars VOP1 showed the highest percentage of non-essential amino acids to thetotal protein content, whilst the percentage of essential amino acids to the total protein content washighest in cowpea cultivars VOP2, VOP3 and VOP7. Highest percentage of branched amino acids tothe total protein content was detected in cowpea cultivars VOP2 and VOP7.

The results of amino acid analysis revealed that cowpea leaves contained both essential andnon-essential amino acids and significant variation in the concentrations was the result of genotypiceffects [47]. Non-essential amino acids, such as serine (Ser), arginine (Arg), glycine (Gly), aspartate(Asp), glutamate (Glu), alanine (Ala), proline (Pro), tyrosine (Tyr), were detected in all cowpea cultivars(Table 4). The Asp and Glu were identified as the predominant non-essential amino acids. Moreover,the cowpea cultivar VOP1 demonstrated the highest concentrations of Asp, followed by VOP4, VOP5and VOP8. Cowpea cultivars VOP3, VOP4, VOP5 and VOP7 had the highest concentration of Glucompared to the other cowpea cultivars (Table 4).

Essential amino acids, histidine (His), threonine (Thr), lysine (Lys), methionine (Met), valine (Val),isoleucine (lle), leucine (Leu), phenylalanine (phe) were detected in all cowpea cultivars (Table 4),in which Leu was found as the predominant essential amino acid. Cowpea accessions VOP3, VOP4,VOP5 and VOP7 contained the highest concentration of Leu, whereas cowpea cultivars VOP3 andVOP4 contained the highest concentration of Phe (Table 4). Cowpea cultivar VOP7 was rich in Lys,followed by VOP4, VOP5 and VOP8. A moderately higher concentration of Val was detected incowpea cultivars VOP3, VOP4, VOP5 and VOP7 (Table 4). Whilst His and Met were detected at lowerconcentrations in all cowpea cultivars, VOP4 and VOP3 contained the highest concentrations of His(Table 4). The trend or pattern in amino acid composition could relate to possible inherent differencesbetween genotypes/cultivars. The more or less similar trends observed regarding the concentrations ofessential amino acids in cowpea cultivars VOP3 and VOP4 probably confirm that these cultivars aregenetically similar compared to the other cultivars. The amount of amino acids in cowpea leaves islower than the cowpea grains [39], which supports the findings of the present study.

The daily requirement of essential amino acid intake from cowpea cultivars VOP3 or VOP4 wascalculated based on the FAO/WHO/UNU [46] guidelines, suggesting that a 110.95 g serving portion ofcowpea accessions VOP3 or VOP4 is able to fulfil the daily requirement of Phe, Leu and lle for adultswith 70 kg body weight. Similarly, 20.95 g of serving portion of leaves of the corresponding cowpeacultivars fulfils the daily requirement of Thr for adults (70 kg body weight). Thus, cowpea cultivarsVOP3 and VOP4 showed potential to fulfil the daily requirements of some essential amino acids.

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Table 4. Non-essential and essential amino acids in cowpea cultivars.

Non-Essential Amino Acids (g/100 g DW)

Cowpea cultivars Ser Arg Gly Asp Glu Ala Pro Try

VOP1 1.38 ± 0.41b 1.92 ± 0.20c 1.33 ± 0.26c 5.96 ± 0.31a 3.28 ± 0.12bc 1.44 ± 0.34c 1.32 ± 0.25b 1.30 ± 0.14bVOP2 1.29 ± 0.23b 1.96 ± 0.12c 1.28 ± 0.17c 3.46 ± 0.10c 3.07 ± 0.38c 1.42 ± 0.21c 1.13 ± 0.34c 1.20 ± 0.21bVOP3 1.60 ± 0.30a 2.42 ± 0.25a 1.71 ± 0.11a 3.49 ± 0.26c 3.67 ± 0.40a 1.56 ± 0.16b 1.43 ± 0.31a 1.91 ± 0.30aVOP4 1.70 ± 0.34a 2.52 ± 0.20a 1.62 ± 0.20a 4.80 ± 0.21b 3.47 ± 0.51b 1.58 ± 0.24b 1.45 ± 0.11a 2.01 ± 0.42aVOP5 1.42 ± 0.20b 2.20 ± 0.31b 1.53 ± 0.15b 4.96 ± 0.18b 3.88 ± 0.12a 1.64 ± 0.10ab 1.46 ± 0.20a 1.24 ± 0.20bVOP7 1.40 ± 0.42b 1.82 ± 0.40c 1.50 ± 0.31b 3.92 ± 0.11c 3.73 ± 0.11a 1.74 ± 0.50a 1.44 ± 0.23a 1.27 ± 0.10bVOP8 1.37 ± 0.21b 1.80 ± 0.30c 1.36 ± 0.20bc 4.63 ± 0.09b 3.42 ± 0.27b 1.53 ± 0.34bc 1.25 ± 0.12b 1.36 ± 0.20b

Essential amino acids (g/100 g DW)

His Thr Lys Met Val lle Leu phe

VOP1 0.50 ± 0.16b 1.41 ± 0.27bc 1.75 ± 0.20c 0.31 ± 0.70 ** 1.26 ± 0.51c 1.41 ± 0.32c 2.13 ± 0.32cd 1.60 ± 0.10bVOP2 0.51 ± 0.29b 1.31 ± 0.10c 1.71 ± 0.17c 0.30 ± 0.50 1.28 ± 0.41c 1.22 ± 0.17d 2.05 ± 0.17d 1.41 ± 0.20cVOP3 0.72 ± 0.20a 1.67 ± 0.30a 1.51 ± 0.41d 0.32 ± 0.14 1.56 ± 0.12a 1.54 ± 0.12b 2.46 ± 0.15a 2.45 ± 0.23aVOP4 0.66 ± 0.18a 1.65 ± 0.25a 1.39 ± 0.32e 0.25 ± 0.28 1.55 ± 0.30a 1.66 ± 0.25a 2.44 ± 0.31ab 2.46 ± 0.16aVOP5 0.45 ± 0.10b 1.39 ± 0.60bc 1.91 ± 0.50b 0.29 ± 0.12 1.54 ± 0.18a 1.58 ± 0.50ab 2.35 ± 0.40b 1.48 ± 0.17cVOP7 0.46 ± 0.38b 1.49 ± 0.10b 2.04 ± 0.27a 0.38 ± 0.32 1.53 ± 0.10a 1.41 ± 0.12c 2.47 ± 0.51a 1.65 ± 0.32bVOP8 0.51 ± 0.41b 1.40 ± 0.31bc 1.92 ± 0.15b 0.27 ± 0.41 1.39 ± 0.21b 1.30 ± 0.10d 2.19 ± 0.30c 1.49 ± 0.12b

DW—dry weight. Data present mean and standard deviation (n = 3). Different letters at the same column indicate significant differences at (p < 0.05). ** are not significant.

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4. Conclusions

This study illustrated the carotenoid and amino acid profile in different cowpea accessions grownin Africa. Leaves of cowpea cultivar VOP2 are a rich source of lutein. Concentration of identifiedphenolic compounds varied among the cowpea cultivars. Chemomertic analysis indicated, based onthe phenolic metabolites, that cowpea accession VOP1 significantly differed from the rest. The Pearsoncorrelation test results showed that gentisic acid-5-O-glucoside, quercetin 3-(2G-xylosylrutinoside) andquercetin 3-glucosyl-(1->2)-galactoside in cowpea cultivars VOP1 and VOP4 might be most responsiblefor the observed in vitro α- amylase and α-glucosidase activities. Leaf extracts of cowpea cultivarsVOP1 and VOP4 enhanced the upregulation of glucose transporter GLUT4 gene and showed similaranti-hyperglycaemic effects to insulin. This study further confirms the relationship between cowpealeaf phytochemicals and glucose metabolism/diabetes.

Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/9/1285/s1,Figure S1: One representative UPLC–Q-TOF/MS chromatogram illustrating the predominant phenolic compoundsin the leaves of cowpea cultivars VOP1, VOP2, VOP3, VOP4, VOP5, VOP7 and VOP8, Figure S2: MS spectra,Figure S3: Score plot of Principal component analysis (unsupervised) based on UPLC–Q-TOF/MS spectra of theleaves of different cowpea cultivars, Figure S4: Histogram illustrating quantitative differentiation of biomarkersbetween the Cowpea cultivar VOP1 and other cowpea cultivarsVOP2, VOP3, VOP4, VOP5, VOP7, and VOP8,Figure S5: Percentage cell viability of C2C12 myoblast cell lines exposed to three different concentrations of leafextracts of cowpea cultivars, Figure 6A: Representative UV chromatogram of carotenoids in cowpea leaves at @450nm, Figure 6B: Representative TIC and mass features of individual carotenoid compounds detected in cowpealeaves, Table S1. Primer sequences used to amplify, the GLUT4 and GAPDH cDNA, Table S2: Pearson’s correlationcoefficients between targeted phenolic components and in vitro antioxidant (FRAP), α-glucosidase and α-amylaseactivities, Table S3 Characterization of carotenoid compounds detected in cowpea accessions by LC–APCI-MSscanning at positive mode.

Author Contributions: M.R.M.—PhD student performed the analysis, wrote the first draft, A.D.T.P.—Postdocoralresearcher executed the carotenoid analysis, data validation, visualization, J.L.S.—responsible for antidiabeticassays and data presentation, Y.S.—co supervisor, guidance, methodology, editing, D.S.—Grant holder,conceptualization, project administration, data validation, editing. All authors have read and agreed to thepublished version of the manuscript.

Funding: The financial support from the Department of Science and Innovation, Government of South Africa andthe National Research Foundation (Grant number 98352) for Phytochemical Food Network to Improve NutritionalQuality for Consumers is greatly acknowledged.

Conflicts of Interest: The authors declare no conflict of interest.

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